Assessment transcallosal Diaschisis in a model of focal cerebral ischemia in rats.

OBJECTIVE
To evaluate transcallosal changes after a local ischemic injury in rats by using the monoclonal marker anti-NeuN (Mouse anti-neuronal nuclei).


METHODS
Twenty-eight adult, male, Wistar rats were subjected to focal injury in the right hemisphere. The technique used was the experimental model of focal ischemic injury through intraluminal suture of the middle cerebral artery. Analyses were made for the five groups: after the lesion (control), at 24 h, 96 h, 10 days and 20 days. Exofocal neuronal damage was inferred from neuronal immunoreactivity changes to NeuN.


RESULTS
In the cortex contralateral to the lesion, immunoreactivity was diminished. This finding was most notable in the supra-granular sheets 24 h post ischemia. After 96 h, there was a generalized diminishment of the inmmunoreactivity in the supra and infra-granular sheets. At 10 and 20 days, the tissue recovered some immunoreactivity to NeuN, but there were some changes in the VI layer.


CONCLUSION
The immunoreactive changes to NeuN support the process of inter-hemispheric diaschisis. Changes in immunoreactivity could indicate metabolic stress secondary to the disruption in connectivity to the site of lesion.


Introduction
Cerebrovascular disease (CVD) is a public health problem in the world. It is the leading cause of permanent disability in adults and the third leading cause of death in developed countries 1 . One out of six people suffers stroke (CVA), resulting from CVD during their lifetime, and about 30% have severe or permanent dysfunctions that make them dependent on others. According to the compromised cerebral territory, alterations can be motor, sensory, or cognitive mnemonic, among others, although generally there is a mix commitment 2 . Alterations may change over time due to exo-focal changes from injury caused by a plasticity process of the nervous system called diaschisis [3][4][5] .
Strokes can be divided into ischemic, by decreased blood flow in specific brain area; or hemorrhagic, due to intracranial arterial rupture. Clinical practice says that 85% of strokes are ischemic; one of the most common is focal ischemia by occlusion of the middle cerebral artery (MCA) 6 .
Cerebral ischemia is a pathophysiological condition characterized by transient or permanent, focal or global decrease of blood flow in the brain, which causes different patterns of tissue dysfunction according to the severity and duration of the ischemia 7 . When the flow of oxygen and glucose falls below 10 mL/100g/min, there is loss of function of the cell membrane by metabolic depletion of ATP pumps, the ion potential is lost, and cell swelling occurs due to increased intracellular sodium, leading in minutes to tissue necrosis and triggering inflammatory processes 8 . There is also an increase in the electrical activity of cell membranes at the edge of the focus, due to increased extracellular glutamate and potassium. This increases the excitability in cells, increases metabolic rate, and leads to the growth of the infarcted area. The directly affected area is called an ischemic focus, and it is the region showing the first consequences of the lesion 9-10 .
Next to the ischemic core, it is established the penumbra, an area with low perfusion in which a residual flow persists, able to keep alive neurons. However, these neurons are dysfunctional due to metabolic stress. Because necrosis has not been established in the penumbra, it is an area susceptible of recovery; however, if an adequate reperfusion is not present, necrosis and stabilization of clinical deficit occur [7][8][9][10][11] . The penumbra is one of the therapeutic targets to reduce the consequences of ischemia, since it is where the needed angiogenesis processes are triggered to restore blood flow, and apparently, to stimulate endogenous neurogenesis 12 . However, there are zones of the penumbra area that are not restored, and that despite normalization of blood flow, undergo apoptotic cell death, days or months after the ischemia 13 .
The ischemic cascade not only affects the focus and penumbra, but also exo-focal areas ipsilateral and contralateral to the lesion, and other structures such as the cerebellum, the thalamus, the substantia nigra, and the hippocampus. These areas and structures are disrupted due to their anatomical or functional connections with the ischemic focus and the penumbra area [14][15][16] . This phenomenon is not only related to pathological processes, but also with the reorganization of the nervous system and functional recovery 17 , and it is seen in other disorders such as tumors, epilepsy and traumatic injuries 18 .
There is evidence of transcallosal or contralateral diaschisis, due to the finding of bilateral brain disorders after unilateral lesions. Taking as stimulus the injury, the reorganization of the unaffected hemisphere is presented by disinhibition of commissural pathways and the relative increase of subcortical neuronal excitability, especially thalamic, probably as a resource to counteract the loss of contralateral function [19][20] . In this sense, the contralateral exo-focal alterations may be associated with brain plasticity, mechanisms of neuronal reorganization, and compensatory and adaptive processes [21][22][23][24][25] . It has been identified that, if it is performed ablation of the corpus callosum, the contralateral effects are reduced or do not occur, and that these changes create tolerance to future ischemic events 24-26 .
In this aspect, it has been described the activation of over 80 genes in the ipsilateral and contralateral cerebral cortex, hippocampus, striatum and thalamus, which relate to circulatory and metabolic modifications 27 . It has also been shown dendritic arborization and axonal growth in the contralateral hemisphere, expression of neurotrophins with motor and sensory recovery, related to the organization of the pyramidal system after ischemia 28 . In addition, there is evidence of neurogenesis and synaptogenesis processes in some areas, and adaptive changes in the entorhinal cortex, among others 24-25,29 .
Although it is not clear the mechanism of transcortical diaschisis 30 , clinically, it is known that the contralateral alterations contribute to neurological deficit and heterogeneity of signs and symptoms in the mid and long term, such as cognitive deficits, loss of memory, aphasia and episodic amnesia [31][32] . However, few proposals are known to address this aspect of the cerebral damage 2-4 . From the above, it is concluded that it is important to study the contralateral hemisphere after ischemic brain injury as a contribution to theories of recovery and reorganization of brain functions. This is of clinical interest because, often, dysfunction and recovery are not directly related to the area and size of the lesion 32 . Learning about the pathophysiology of ischemia and transcortical changes is fundamental to understand the restoration processes of the systems affected by the injury, and consequently, to determine the most effective type of treatment.
The aim of this research was the descriptive characterization of contralateral cortical changes in an experimental model of focal cerebral ischemia to get evidence of cell reorganization, in order to understand the pathophysiological changes. It was used a monoclonal antibody anti-NeuN, specific for neurons (Neuronal Nuclei MAB377), with which, besides the nucleus, it is possible to mark the soma and the proximal processes of neurons. The marking process does not include glial cells or vascular components 34 , unlike conventional histological methods that can eventually produce "noise" when observing cells. Within the nucleus, NeuN reactivity is mainly found in areas where there is low chromatin density, and it is largely excluded from areas containing densely packed DNA. The enzymatic dephosphorylation suppresses the immunoreactivity to NeuN, confirming that this is indeed a phosphoprotein, and it states that the antibody binding depends on the phosphorylation state 9 . It is clear that protein phosphorylation circumstances are significantly altered in ischemia. The NeuN has been identified with the Fox-3 protein, which like other proteins of this type, regulates mRNA splicing 35 .
In pathological conditions such as cerebral ischemia, the decrease in quantifying NeuN cells may indicate neuronal loss due to necrosis 34 . In addition, it has been described a correlation among apoptotic cells, positive tunnel and immunoreactivity to NeuN with apoptotic cell death 36 . However, it has been established that the decrease in immunoreactivity to NeuN may be related to protein depletion or reduced immunoreactivity; changes that are not necessarily related to cell death, but with cellular metabolic stress 37 . If we consider that the transcallosal diaschisis corresponds to a phenomenon of cellular stress, assessing NeuN immunoreactivity in the cortex contralateral to the lesion can become a sensitive marker of exo-focal phenomena.

Materials and Methods
Twenty eight Wistar adult male rats were operated. The specimens weighted between 240 and 320 g (average 280 g), and were aged between 120 and 180 days. It was followed the protocol to avoid unnecessary suffering.
Microsurgical procedure After a fasting period of 6 h, the experimental specimens were anesthetized with intraperitoneal ketamine hydrochloride (40 mg/kg) and atropine (0.1 mg/kg). The cerebral ischemia technique applied in this paper was developed by Zea et al. 38 , and perfected by Belayeb et al 39 . Under a surgical microscope, a longitudinal incision was made in the midline of the neck. The common carotid artery and the external carotid artery (ECA) were located and dissected, being the latter cut, thus leaving free the ECA and linked at its end. A 4-0 nylon monofilament segment of 3.5 cm in length was introduced by the bottom of the corresponding ECA sac to a length of 17.5 mm and then fixed. After 90 min, the nylon was pulled through the surgical wound, and its end was left housed at the bottom of the ECA sac to start reperfusion. The four specimens of the control group underwent the described procedure, except that the 4-0 nylon monofilament was not introduced until the level of the middle cerebral artery (MCA). During the surgical procedure, the cardiorespiratory function and the rectal temperature were regularly evaluated. Temperature was maintained at 37° C by a heat lamp. Five groups were established, one control group with four specimens, and the remaining four with survival times at 24 h, 96 h, 10 days and 20 days; each one with six specimens.

Immunohistochemistry
At the corresponding time, the specimens of each group were anesthetized and trans-cardially perfused through the left ventricle with 200 mL of 0.9% saline, followed by 200 mL of a mixture of paraformaldehyde (4.0%), lysine and sodium periodate in 0.1 M phosphate buffer (pH 7.4). Then, the brains were removed from the braincase and left in the fixative. Later, 10 coronal sections of 30 µm thickness were performed in the vibratome of liquid medium; the cuts were made in accordance with the coordinates of the Atlas of Paxinos and Watson 40 in a sequential anteroposterior way, starting from the interaural point 10.7 with bregma 1.7, each mm to the interaural 3.7 point with bregma -5.3. The sections were incubated with the primary antibody NeuN (Neuronal Nuclei -MAB377 Chemicon) for 8 h; then, they were washed in phosphate buffer 10 mM (pH 7.4) and made react with secondary antibody using the avidin biotin kit (Vectastain), and then were revealed using diamino benzidine contrasted with nickel (ABC kit vector laboratories). The cuts, selected of the samples as control for the immunohistochemical process, suffered the same procedure described above, except for incubation with the primary antibody.
The cuts of the different experiment times were stained with 2,3,5-Triphenyltetrazolium chloride (TTC) to observe the infarcted area; for this type of vital staining, one of the subjects in each group was not perfused, it was deeply anaesthetized, and it was decapitated; then, the brain was rapidly removed from the braincase. The brains were cut coronally every 2 mm from the frontal pole using a brain matrix (Stoelting). The brain sections were immersed in a saline phosphate buffer solution (PBS) 0.1M pH 7.2 containing 0.5% TTC during 30 min at 37 o C. Finally, the stained sections were fixed in 4% formalin and kept at 4 o C. Six sections per brain were photographed in their anterior and posterior aspects, and the ischemic areas were manually measured in a blind way. Morphometric and volumetric analysis implied to correct the error caused by edema, for which it was used the formula [(normal hemisphere area / infarcted hemisphere area) x infarct area] 41 . All experimental specimens showed lesion in an area proportional to approximate coronal section in Bregma 2.04 mm of 39.4 ± 4.2%. There were no major differences among the specimens, and variability in size among the groups was very low. The volume of ischemic damage was calculated by integrating the area of the corrected lesion of the different coronal cuts with the anteroposterior coordinate 42 ; thus, the result is expressed in cubic millimeters of lesion; it was observed a substantial homogeneity in the different specimens 162.5 ± 17.5 mm 3 .
The evaluation of the processed cuts by NeuN immunohistochemistry was performed in a qualitatively semistructured way, since each of the researchers independently examined the plates at 10x and 40x with specimen cuts in the control group and the experimental ones, focusing on cuts corresponding to the frontoparietal cortex, this was systematically done following an encoded sequence with a previously established order, and without knowledge of the type of the specimen that was being observed. Prior to this procedure, it was performed an assessment and training of all researchers to ensure the greatest possible objectivity in the observations. It began with a global observation of the sheets at 10x, and then a review sheet by sheet at 40x from I to VI, paying attention to the degree of immunoreactivity of the neurons to NeuN (immunoreactivity intensity similar to control v/s immunoreactivity intensity lower than control -neuronal morphology similar to control v/s neuronal morphology different to control). Each of the descriptions was recorded in the same type of format to be collected by the principal investigator, images for analysis of results were selected when the coincidence of the observation was 80% or more. To obtain the pictures, researchers were asked to select three patterns considered as representative of each change observed by them as relevant; from this selection, in turn, the lead researcher took random pictures to illustrate the publication, and for the final analysis.
On the other hand, changes were quantified in the number of positive NeuN cells in lamina VI: three areas of similar size for each of the specimens were demarcated, photographed and processed in black and white to 50% in the image format of the program World of Office, the images were printed and neurons were manually counted. Each of researchers sent the results and they were integrated by a simple summation to define the proportion of neurons relative to the control.

Ethics
The state of control of the 28 subjects in all phases of the research was conducted using a monitoring protocol that establishes criteria to avoid unnecessary suffering, especially after surgery.

Results
There were processed from the control group, six specimens. From the experimental groups of 24 h, 4 specimens; 96 h, 5 specimens; 10 days, five specimens; and 20 days, 4 specimens.
In this study the changes in the region contralateral to the lesion of the frontoparietal cerebral cortex were evaluated qualitatively. The results were obtained based on the data provided by the experimental groups, confronted with the control group, and descriptive analysis of changes in the immunoreactivity of neurons to NeuN. In all specimens subjected to ischemia and reperfusion, it was observed frontoparietal lesion in the territory of the MCA ipsilateral to the lesion, which includes loss of anatomical and histological features with degrees of neuronal damage ranging from obvious injury at 24 h, up to total loss of tissue with the formation of cystic areas (Fig. 1), as we have reported in other studies 4,9,33 .
At 24 h, in the contralateral area, there were changes in neurons with decreased immunoreactivity to NeuN, compared with controls ( Fig. 2 C and D); these changes are remarkable in supragranular layers. At 96 h post-ischemia, in the cortex of the hemisphere contralateral to the lesion, it is characteristic the decrease of cell immunoreactivity in supra and infragranular layers. In addition, it is appreciated cell nuclei with fractionation and consolidation with morphologic appearance resembling the nuclear fragmentation observed in apoptotic cell death (Fig. 2E and F). At 96 h, it can be seen transition areas from an organized tissue, to one where the cyto-architecture appearance is lost, especially in layer VI, which includes areas of disruption in the continuity of the layer and apparent neuronal loss (Fig. 3C).
Ten days post-ischemia in the contralateral hemisphere to the lesion, it can be seen unchanged cortical layers in their immunoreactivity, when they are compared with control ( Fig. 2 G  and H). At 20 days post-ischemia, the finding is indistinguishable from those found in the specimens at 10 days, which consist in an immunoreactivity to NeuN similar to controls; different from that observed in samples at 24 and 96 h.
An interesting observation was that 60% of subjects had alterations in the morphology of the layer VI (Fig. 3), changes that were detected in a more evident way at 96 h post-ischemia (Fig. 3 C), and that continued being seen until 10 days (Fig 3 D.) and 20 days (Fig 3 E.). It was observed in this layer impairing of the tissue cyto-architecture and a progressive decrease and loss of neuronal  immunoreactivity: relative to quantification of neurons in the controls that we defined as a proportion of 100%, the number of immunoreactive neurons decreased to 95.5% and 77.3% at 24 and 96 h, respectively; at 10 and 20 days, the amount of immunoreactive neurons corresponds to 63.6% and 59.1% respectively. Panoramic view at 10x magnification, it is seen the loss in patches in some sectors of the layer VI (Fig. 3F).

Discussion
The analysis of changes in neurons of the hemisphere contralateral to focal ischemic injury has been little evaluated by using specific markers for neuronal population [43][44][45] . Nagasawa and Kogure 46 found no histological abnormalities in the contralateral hemisphere after focal cerebral ischemia in an experimental model similar to ours. They used Nissl staining and hematoxylin, which are not specific for neuronal population 42 . On the other hand, it has been found differences in susceptibility to stroke among different strains of rats 47 , which could be related to the heterogeneity of the results of various investigations, especially regarding contralateral exo-focal changes.
In this study, the intensity of the immunoreactivity was considered as a pathophysiological measure of ischemic brain damage, as it has been noted in other works 37,48 . The most significant changes are in dorsolateral areas in both hemispheres, regions that suffer in this model increased ipsilateral neuronal loss and changes in immunoreactivity of neurons in the contralateral cortex. In the frontoparietal region ipsilateral to the lesion, varying degrees of neuronal damage were observed, ranging from obvious necrosis at 24 h, up to total loss of tissue with formation of cystic areas, as it has been reported in other studies 4,9,49 .
With regard to exo-focal changes in cortex contralateral to lesion, our study has three aspects to highlight. First, at 24 h after ischemia, decreased immunoreactivity to NeuN is evidenced in the supra-granular layers of the frontoparietal cortex contralateral to injury; at 96 h, changes are evident in both supra-granular and infra-granular layers. It is noticeable the fact that some neurons at 96 h exhibit immunoreactivity that account for an apparent fragmentation of nuclei, hitherto not described with NeuN (Fig.  2 F). It is important to note that previous studies have established a co-localization of neurons with reduced immunoreactivity to NeuN, and positive tunnel test 37 .
Second, 10 and 20 days after ischemia, the immunoreactivity to NeuN, previously attenuated, is reset in supra-granular and infragranular areas, but an alteration of residual appearance in layer VI is evident (Fig. 3), because it remains from 96 h until 20 days posterior to the ischemic injury. Previously, there were described processes of cortical plasticity related with thalamus-cortical connections, since the response of the cortex could be related to afferents coming from the thalamus to the layer VI 20,50 . Then, it arises the question that apart from the signals of de-afferentation of the ischemic focus through the corpus callosum, thalamic afferents would have an effect on cortical reorganization 17,19 .
Third, it has been noted that the mechanisms of transcallosal exo-focal changes involve commissural connections and synapsis modulation 4,15,51 . Hyper-excitability has been observed in the contralateral hemisphere, as well as changes in metabolism that could be related to these transcallosal exo-focal changes 52 . The decrease in the activity of GABAergic neurons in the injured hemisphere and increased glutamatergic activity caused by ischemia can translate into overdrive in the not-injured hemisphere 26 . In this way, it could be explained the reorganization of motor functions after a CVA 51,52 . It has been described that in rats subjected to focal ischemia, the motor cortex in the non-ischemic hemisphere undergoes a process of reduction of dendritic spines, followed by arborization in the infra-granular layers. At the peak of dendritic growth, 18 days after ischemia, the synaptic density in infra-granular layers and spine density is normal 16 . This is a descriptive study of immunoreactivity to NeuN in the cortex contralateral to lesion. While it allows us to observe a sequence of events related to the intensity of the immunohistochemical reaction and morphological findings, the results should be expanded in subsequent studies, in which optical densities and quantification of neurons can be assessed. Other studies are also missing, that expand our insight about the role of NeuN antibody as a marker of cellular stress and potential marker of cellular death.

Conclusion
This study descriptively shows changes in cerebral cortex contralateral to lesion after transient focal ischemia in the territory of the middle cerebral artery in an experimental model in rats. Immunoreactivity to NeuN is taken as the primary means of analysis. So far, no studies are known of transcallosal diaschisis with observation of such marker. It was observed specific variation in the intensity of the immunoreactivity to NeuN as a function of time from 24 h to 20 days, showing a sub-chronic response in supra-granular areas, and then in infra-granular ones. It is remarkable to observe changes in layer VI that remain from 96 h up to 20 days. In functional terms, the significance of changes in neuronal immunoreactivity to NeuN could be associated with adaptive processes of contralateral cell reorganization after Figure 3. Images representative of immunoreactivity to NeuN in layer VI of frontoparietal cortex contralateral to ischemic injury, in an experimental model of focal cerebral ischemia of the middle cerebral artery. A control; B to F, changes in layer VI remarkable in the course of time; it is indicated, with boxes, the areas with altered immunoreactivity to NeuN, and apparent decrease of cellularity in F with panoramic view at 10x, it is seen loss in patches in some sectors of the layer VI. ischemic event, in which cortico-cortical and cortico-thalamic projections would participate, as well as local circuits.
Financing: Pontificia Universidad Javeriana, Cali, Colombia (Code 00003579) Neuroscience and Neuropsychology Line from the Group of Measurement and Psychological Assessment. Project "Reorganization on exo-focal regions (substantia nigra reticulata and dendritic fascicles in the frontoparietal cortex) for experimental transient focal ischemic injury in rats". For the information processing and work synthesis, we had support from resources of Valle del Lili Foundation and ICESI University of Cali, Colombia. We also had resources from the Innovation and Science Foundation for Colombia FICC through the Center for Innovation Research and development IDRC Line of Research in Experimental Neurosciences, Project "Brain Injury and Neurorehabilitation", providing human talent and micro-photographic record.
Knowledge: Thanks to the Animal Ethics Committee of Universidad Libre of Cali, which evaluated this work.